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Key gene TcSBP5 regulating salt tolerance of tamarix chinensis and application of key gene TcSBP5

A key gene, salt tolerance technology, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of reducing the germination rate of salt tolerance

Active Publication Date: 2019-07-09
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the SBP transcription factors of Tamarix plants. The cloning and development of Tamarix SBP transcription factors will not only help to elucidate the molecular regulation mechanism of high salt tolerance in halophytes, but also provide important information for the screening of important salt tolerance genes and stress resistance. Genetic breeding provides a theoretical basis and molecular tools, and has important application value for the utilization of saline-alkali land and the improvement of comprehensive agricultural production capacity

Method used

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  • Key gene TcSBP5 regulating salt tolerance of tamarix chinensis and application of key gene TcSBP5
  • Key gene TcSBP5 regulating salt tolerance of tamarix chinensis and application of key gene TcSBP5
  • Key gene TcSBP5 regulating salt tolerance of tamarix chinensis and application of key gene TcSBP5

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Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1: clone TcSBP5 gene by RACE technology

[0022] Based on the TcSBP5 sequence of Tamarix RNA-seq data, 5′ and 3′ RACE primers were designed, two specific products were amplified by nested PCR, sequenced by T-vector cloning, and the sequencing results were spliced ​​by overlapping regions to obtain the full length of cNDA .

[0023] details as follows:

[0024] I. Primer Design

[0025] The 3'RACE forward primer is:

[0026] Outer Primer F:

[0027] Inner Primer F:

[0028] The 3'RACE reverse primer is:

[0029] Outer Primer R:

[0030]

[0031] Inner Primer R:

[0032] The 5′RACE forward primer is

[0033] Outer Primer F:

[0034]

[0035] Inner Primer F:

[0036] The 5′RACE reverse primer is:

[0037] Outer Primer R:

[0038] Inner Primer R:

[0039] II.3'RACE reaction process:

[0040] (1) For reverse transcription, add the following components to an RNase-free centrifuge tube placed on ice: 1 μg Total RNA (the plant material...

Embodiment 2

[0074] Embodiment 2: TcSBP5 gene plant expression vector construction

[0075] The overexpression vector of TcSBP5 gene was constructed by pathway cloning technology. Using specific PCR primers (TcSBP5 ORF primers in Example 1), PCR amplification was performed using cDNA as a template, and the TcSBP5 gene ORF was constructed into an entry vector. The entry vector was pCRTM8 / GW / TOPOTM vector (Invitrogen). The reaction system is: Fresh PCR product (purified) 10-20ng; Salt solution 1μL; pCRTM8 / GW / TOPOTM vector 1μL; add sterile ddH2O to make up 6μL. The reaction procedure is: stand at room temperature for 30 minutes.

[0076] Pick positive clones from the screening culture plate for sequencing verification, and perform LR reaction with the positive entry vector and the plant expression vector PBI121GW. Vector plasmid such as figure 1shown. The reaction system is: entry vector 100ng; PBI121GW vector (100ng / μL) 1.5μL; LR Clonase II enzyme mix 2μL; add TE (pH 8.0) to make up 10μ...

Embodiment 3

[0077] Example 3: Genetic transformation of the TcSBP5 gene

[0078] The constructed PBI121GW-TcSBP5 overexpression vector was transformed into Agrobacterium strain EHA105 by liquid nitrogen freeze-thaw method, and the TcSBP5 gene was transformed into Arabidopsis by Agrobacterium tidbits infection method. The obtained positive Arabidopsis seeds were tested for germination rate and plant phenotype observation on MS solid medium containing 0.3% NaCl (51 mM). figure 2 PCR detection of TcSBP5 insert fragments for 10 successfully transformed transgenic Arabidopsis; image 3 This is the overall morphology comparison between the transgenic Arabidopsis overexpressing TcSBP5 and the wild type. The growth substrate is MS solid medium containing 0.3% NaCl (51mM). After 14 days of growth in Arabidopsis 0.3% NaCl medium, it was transferred to NaCl-free MS medium for desalination treatment for 14 days, 3 was wild-type Arabidopsis 0.3% NaCl medium for 28 days, and 4 was wild-type Arabidops...

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Abstract

The invention discloses a key gene TcSBP5 regulating the salt tolerance of tamarix chinensis. The nucleotide sequence of the key gene TcSBP5 is shown in SEQ ID No.1. A protein product expressed by theTcSBP5 is a tamarix chinensis SBP transcription factor of which the nucleotide sequence is shown in SEQ ID No.2. The key gene TcSBP5 regulating the salt tolerance of the tamarix chinensis has the advantages that arabidopsis thaliana is converted to obtain arabidopsis thaliana of an overexpressed TcSBP5 gene, the salt-tolerant germination rate of seeds of the arabidopsis thaliana is significantlyreduced, the salt-tolerant physiological index of a plant of the arabidopsis thaliana is significantly reduced, the arabidopsis thaliana shows the typical salt-sensitive phenotype of growth inhibitionon the whole, and it is shown that the gene is an important key factor regulating the plant salt tolerance and has important application value in the field of salt-tolerant breeding of trees.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a key gene TcSBP5 for regulating the salt tolerance of Tamarix tamarix and its application. Background technique [0002] According to the statistics of the Food and Agriculture Organization of the United Nations, 6.5% of the total land area is saline-alkali land that cannot be cultivated. The area of ​​saline-alkali land in my country is about 200,000 square kilometers. The high-salt stress in saline-alkali land directly affects crop yields. Making full use of the saline-alkali land through stress-resistant breeding methods is beneficial to increase Crop production and solving the food crisis are of great significance. Although some general salt-tolerance mechanisms have been elucidated in model species such as Arabidopsis and rice, many studies on salt-tolerance genes have shown that the effect of improving the salt-tolerance of transgenic plants usi...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/8273
Inventor 胥猛王建文叶友菊陈彩慧樊航
Owner NANJING FORESTRY UNIV
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